Encephalopathy due to elevated ammonia levels is a common and costly clinical condition associated either with congenital deficiency of hepatic urea cycle enzymes, with acquired liver disease and with a variety of other conditions. Despite a long recognition that elevated ammonia levels can lead to impaired neurologic functioning, the exact mechanism through which this occurs remains incompletely understood. Moreover, treatment of hyperammonemic encephalopathy has been limited by a lack of knowledge of the specific CNS protein(s) with which ammonia directly interacts. The broad, long-term objective of this project is to examine the hypothesis that Rh C Glycoprotein (RhCG) is a central nervous system (CNS) ammonia 'sensor.' We hypothesize that RhCG protein is expressed in specific CNS neuron populations, that ammonia-stimulation of RhCG is coupled to specific intracellular signaling pathways, most likely including MAP kinase, and that RhCG expression is regulated by specific physiology/pathophysiologic stimuli. We will examine this hypothesis with two specific aims. In the first, we will determine whether ammonia's stimulation of RhCG, or, possibly the related proteins, Rh A Glycoprotein (RhAG) or Rh B Glycoprotein (RhBG), activates specific intracellular signaling pathways in cultured neurons. We will use primary neuronal cultures, and will use RNA interference techniques to inhibit RhCG expression to show specificity of response. In parallel, we will determine whether RhCG can function as an ammonia sensor by determining whether it can complement the pseudohyphal transformation-defect of Amep2-Amep2 S. cerevisiae. To examine the second aim, we will determine whether cecal ligation and puncture-induced sepsis increases CNS expression of either RhCG, or of RhAG or RhBG. These studies will combine immunohistochemical analyses of cellular protein expression patterns with quantitative analyses of protein and mRNA expression with immunoblot and real-time RT-PCR, respectively. These studies fit the purpose of the R21 mechanism in two different manners. These studies will provide pilot data to assess the feasibility of a novel avenue of investigation into the role of RhCG, or related proteins, as CNS ammonia 'sensors.' Second, while these studies are admittedly high risk, their results could lead to a breakthrough in the field of hyperammonemic and hepatic encephalopathy.